Sema4D silencing increases the sensitivity of nivolumab to B16-F10 resistant melanoma via inhibiting the PI3K/AKT signaling pathway

Melanoma is a common skin tumor that causes a high rate of mortality, especially in Europe, North America and Oceania. Immunosuppressants such as anti-PD-1 have been used in the treatment of malignant melanoma, however, nearly 60% of patients do not respond to these treatments. Sema4D, also called CD100, is expressed in T cells and tumor tissues. Sema4D and its receptor, Plexin-B1, play crucial roles in the process of immune regulation, angiogenesis, and tumor progression. The role of Sema4D in melanoma with anti-PD-1 resistance is poorly understood. Through a combination of molecular biology techniques and in silico analysis, the role of Sema4D in improving anti-PD-L1 sensitivity in melanoma was explored. The results showed that the expression of Sema4D, Plexin-B1 and PD-L1 was significantly increased in B16-F10R cells. Sema4D knockdown synergizes with anti-PD-1 treatment, cell viability, cell invasion and migration were significantly decreased, while the apoptosis was increased, the growth of tumors on the mice was also inhibited. Mechanistically, bioinformatics analysis revealed that Sema4D is involved in the PI3K/AKT signaling pathway; the downregulation of p-PI3K/PI3K and p-AKT/AKT expression were observed in Sema4D knockdown, therefore, nivolumab resistance is related to Sema4D and Sema4D silencing can improve sensitivity to nivolumab via inhibition of the PI3K/AKT signaling pathway.


INTRODUCTION
Melanoma, which is prevalent in Europe and North America, has been on the rise in recent years (Rodriguez-Cerdeira et al., 2017). Melanoma is the most aggressive and fatal skin cancer, which can easily metastasize to the whole body through lymph and blood vessels. If the tumor metastasizes to distant and visceral organs, the 5-year survival rate is only 15-20% (Eggermont, Spatz & Robert, 2014;Huang & Zappasodi, 2022). Surgery is an important treatment for melanoma, chemotherapy such as paclitaxel and cisplatin are also effective, and immunotherapy such as anti-PD-1/PD-L1 also plays an important role in changing the survival rate of melanoma (Miller & Mihm Jr, 2006). Because cutaneous melanoma is often associated with UV exposure and elevated tumor mutational load, it is one of the most immunogenic of all cancer types and one of the most effective for immunotherapy (Melixetian, Pelicci & Lanfrancone, 2022).
Immune checkpoints such as programmed cell death protein-1(PD-1) and programmed death-ligand 1(PD-L1) play an important role in immunotherapy. PD-L1 is distributed in tumor cell and PD-1 is generally distributed in T-cells, when PD-L1 bind to its receptor PD-1, triggers inhibitory signaling to attenuate T cell activity (Sun, Mezzadra & Schumacher, 2018). PD-1 or PD-L1 inhibitors such as nivolumab and pembrolizumab can prevent the binding of PD-1 to PD-L1, thus restoring T cell activity and killing melanoma, non-small lung cancer (NSCLC) and other tumors (Larkin et al., 2015;Ribas et al., 2015;Weber et al., 2015). However, clinical evidence indicated that there are only 30-40% of patients receiving PD-1 or PD-L1 inhibitors could achieve a complete response (Hugo et al., 2016). Furthermore, there are a growing subset of responders acquired resistance within two years after treatment in melanoma (Robert et al., 2015a;Robert et al., 2015b). Therefore, it is an urgent to find new biomarkers with predictive value for melanoma or other solid tumors that are ineffective or resistance in the treatment of PD-1 or PD-L1 inhibitors, and ultimately improve the efficacy of PD-1 or PD-L1 inhibitors.
Semaphorins is involved in regulating tumor growth and metastasis. Especially in the tumor microenvironment, the disturbance between different cell types can control the development and progression of cancer (Lontos et al., 2018;Valentini et al., 2021). Semaphorin 4D (Sema4D) is a member of the semaphorins, which plays a important role in the progress of immunoregulatory, platelet inactivation, angiogenesis stimulation and bone formation regulation (Lontos et al., 2018). Sema4D is upregulated in most tumor tissues, such as prostate, colon, breast, melanoma, head, and neck carcinomas (Liu et al., 2015). As a ligand of Plexin-B1, Sema4D plays important roles in tumor cell proliferation, survival, and migration (Ch'ng & Kumanogoh, 2010).
The phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway is involved in the occurrence and development of melanoma. lncRNA OR3A4, GRB2 and Hey1 can promote melanoma migration and invasion via this signaling pathway (Shayan et al., 2017). When PI3K combines with GRB2, it will be phosphorylated and recruited to the cell membrane, then AKT phosphorylatedand acts on its target genes to regulate a variety of cellular processes (Yu et al., 2006).
In this study, we use a combination of molecular biology techniques and bioinformatics to explore the roles of Sema4D in anti-PD-1 resistance melanoma. We demonstrated for the first time that Sema4D is involved in anti-PD-1 resistance in melanoma. Moreover, Sema4D deficiency can overcome resistance to anti-PD-1 therapy and the mechanism is related to PI3K/AKT signaling pathway.

Cell culture and B16-F10 resistance cell line establishment
293T cells, B16-F10 were purchased by Nanjing Cobioer Biotechnology Co., LTD (Nanjing, China), cultured at 37 • C in 5% CO 2 in RPMI-1640 (Life Technologies, Invitrogen, Canada) with 10% FBS (Gibco, Billings, MT, USA). The combination of high dose shock and gradually increasing dose was applied to establish the cell lines insensitive to nivolumab (anti-PD-1). The B16-F10 cell line in the logarithmic growth phase were cultured with nivolumab (AbMole Bioscience Inc., Shanghai, China) 10 ng/mL and TIL at a 1:5 ratio. After 24 h, the solution was changed, and dead cells were discarded. The remaining cells were then cultured with RPMI-1640 without nivolumab, when the remaining cells have stabilized, then cultured with nivolumab 25 ng/ml and repeat these steps until the cells could grow continuously and stably in the medium with nivolumab 50 ng/mL, and then cultured drug-free for 1 month, B16-F10 resistance cell line (B16-F10R) were obtained. B16-F10R also needs with nivolumab 5 ng/mL to maintain the resistance (Maeda, Lafreniere & Jerry, 1991).

shRNA and expression plasmid transfection
Short hairpin RNA (shRNA) targeting Sema4D and the lentivirus containing this shRNA were designed and synthesized by Genepharm Co. (Shanghai, China), sh-sema4D (5 -GGATGGGACTG TCTATGATGT-3 ) and the control NC-sema4D (5 -TTCTCCGAACGTGTCACGT-3 ). The shuttle plasmid and three other packaging plasmids (pGag/Pol, pRev, pVSV-G) were co-transfected into 293T cells to generate a lentivirus expressing Sema4D shRNA or a control lentivirus expressing shRNA. Cells were inoculated into 6-well plates at 1 × 10 5 and infected with lentivirus while 5 µg/mL of Polybrene was added. The culture media was changed after 24 h of infection, and the infected cells were screened with 1 µg/mL of puromycin for 7 days after the culture media change to obtain stable lentiviral shRNA expressing cell lines for subsequent experimental analysis (Moore et al., 2010).

Sema4D overexpression
B16-F10 cells were treated with RPMI 1640 containing 10% fetal bovine serum culture medium was placed in 5% CO 2 incubator at 37 • C, and logarithmic growth was taken long-term cells were used in the experiment. Construct Sema4D lentivirus overexpression vector LV-Sema4D and negative control LV-NC, these vectors were commissioned by Gemma Pharmaceutical Technology Co., LTD. (Shanghai, China) to synthesize. B16-F10 cell was inoculated in 24-well plates and cultured for 24 h multiplicity of infection, complete culture medium was replaced for further culture 48 h, the expression of Sema4D was detected by RT-qPCR.

MTT
Cell viability was measured by Methyl Thiazolyl Tetrazolium (MTT) assay. MTT was dissolved in DMSO and 0.5 mg/mL MTT diluted in 1 × PBS for 4 h, Turn on Benchmark Plus microplate reader (Bio-Rad, Hercules, CA, USA) and detect absorbance at 570 nm (Algazi et al., 2020).

Cell apoptosis analysis
Cell apoptosis were detected by AnnexinV-AlexaFluor647/PI Kit Apoptosis Assay Kit (Biotech, Minneapolis, MN, USA). 5 × 10 5 B16-F10R cells were seeded in 10-cm culture dishes (Corning, Corning, NY, USA) and incubated overnight. 5 × 10 5 treated cells were collected and resuspended with 500 µL binding buffer. 5 µL Annexin V/AlexaFluor647 were incubate for 5 min at room temperature, then add 10 µL propidium iodide (PI), fluorescent dyes should be protected from light. All operations are performed as per manufacturer instructions.

Wound healing and invasion analysis
B16-F10R cells invasion and migration was analyzed by Matrigel-coated invasion chambers and scratch assays respectively. The initial number of cells cultured was 5 × 10 5 , a sterile 10 µL pipette tip was used to mark a line in the monolayer of each well, the wounds were observed at 0, 24 and 48 h under a microscope (Nikon, Japan). ImageJ software was used to measure the wound areas. Cell free migration area =(cell free area 0 h -cell free area 24h)/cell free area 0 h (Ji et al., 2018). 5 × 10 5 cells were placed into the Matrigel-coated invasion chambers (Corning, USA) to incubation, the upper and lower cultures were separated by polycarbonate membranes, and the cells under study were planted in the chamber, after 48 h, the top of Matrigel and invading cells were fixed and stained with 0.1% crystal violet. The microscope (Nikon, Japan) was used to photograph invading cells, and 3 fields were randomly selected and counted each time.

Data collection
Melanoma gene expression profiling data were extracted from the Cancer Genome Consortium (TCGA) database using the TCGAbiolinks software package. Expression profiles were converted to transcripts per million mapped reads per kilobase exon model (TPM) and normalized by log2.

Weighted co-expression network analysis
To explore the signaling pathways involved in Sema4D, gene co-expression network analysis (WGCNA) was first conducted to determine genes with significant change between highand low-expression of Sema4D through the WGCNA package on R project. Topological overlapping measurement was utilized to identify modules most associated with Sema4D expressions. Correlation between modules and traits is calculated by Spearman correlation analysis.

Protein-protein interaction
To explore the potential functions played by Sema4D in various biological processes, we constructed Sema4D-related protein-protein interaction network by STRING (https://cn.string-db.org/).

Gene set variation analysis
Gene set variation analysis was conducted to reveal the molecular mechanisms under the different Sema4D expression populations in melanoma. The h.all.v7.5.1.symbols.gmt gene set was used to enrichment analysis and was downloaded from MSigDB (https://www.gseamsigdb.org/gsea/index.jsp).

Functional enrichment analysis of Sema4D
To explore the mechanism of action of Sema4D involved in PD-1 inhibitor resistance, enrichment analysis on hub genes was performed. ''ClusterProfiler'' package in R project was utilized to identify the signaling pathways involved in Sema4D within TCGA cohort (which contains 471 melanoma samples).

Statistical analysis
GraphPad Prism V.7.00 software was utilized for statistical analysis. Different statistical methods were selected according to different experimental purposes, compare the differences between two groups, we chose Student's t test, one-way ANOVA was used to compare the differences between the overall differences. P < 0.05 is considered to be statistically significant.

Sema4D downregulation inhibit PD-L1 expression upon anti-PD-1 therapy
We analyzed the correlation of PD-L1 and Sema4D with bioinformatics, it showed that PD-L1 and Sema4D are positively correlated ( Fig. 2A) in TCGA-cohort. Compared with Sema4D-NC group, protein expression of Sema4D in Sema4D-shRNA group was significantly downregulated, it indicated that Sema4D silencing is success (Figs. 2B and 2C). Compared with Sema4D-NC group, mRNA and protein expression of PD-L1 in Sema4D-shRNA group was significantly downregulated after nivolumab treatment, which was consistent with the results of bioinformatics (Figs. 2B, 2D and 2E). Therefore, Sema4D silencing significantly repressed the mRNA and protein expression of PD-L1.

Sema4D repression renders B16-F10R cells sensitive to PD-1 treatment
To explore the relationship between Sema4D downregulation and nivolumab treatment efficacy in B16-F10 and B16-F10R cells, we investigated the cell viability and cell apoptosis. First, we observe the effects of nivolumab on the cell viability of B16-F10 and B16-F10 sensitivity to B16-F10 cells. (Fig. 3C). We further explored the effect of nivolumab on the cell viability of Sema4D knockdown B16-F10R cell lines, the results showed Sema4D downregulation potentiates anti-PD-1 treatment efficacy in B16-F10R cells. Compared with Sema4D-NC group, the cell viability of Sema4D-shRNA group in combination with nivolumab was decreased significantly (p = 0.0186) (Fig. 3D) and rate of apoptosis cell in Sema4D-shRNA group after treatment with anti-PD-1 was notably increased (p = 0.0027) (Fig. 3E). Finally, we tested the effect of nivolumab on Sema4D-knockdown melanoma in mice. In vivo experiment, we transplanted B16-F10R cells in the mice and observed the size and weight of tumors treated with nivolumab, tumor volume and weight were significantly delayed in Sema4D-shRNA group mice as compared to Sema4D-NC group mice (Figs. 3F, 3G and 3H).
(G) Tumor weight of knockdown Sema4D with nivolumab, t -test was used to compare the differences. Compared with Sema4D-NC group, *** is p < 0.001, **** is p < 0.0001. (H) Mouse melanoma specimen. Major marks represent cm on the scale.

Sema4D repression potentiates the effect of anti-PD-1 on cell invasion and migration
The effect of Sema4D repression with anti-PD-1 treatment on B16-F10R cell invasion and migration was analyzed by Matrigel-coated invasion chambers and scratch assay. The results showed that when knockdown of Sema4D, cell migration of Sema4D-shRNA group was decreased (p < 0.0001), the cell-free area between migrating cells from Sema4D-shRNA group was wider than the gap between cells of the Sema4D-NC group (Figs. 4A and 4B). In addition, the number of invaded cells was significantly less in Sema4D-shRNA group in contrast to , these data suggesting that lowering Sema4D could enhance the inhibitory effect of anti-PD-1 on cell migration and invasion.

Downregulation of Sema4D inhibit PI3K/AKT expression
The brown module was determined to be most significantly associated with Sema4D expression (r = + − 0.67, p-value = 1e−62), which contained a total of 778 genes (Figs. 5A-5B). Gene set variation analysis (GSVA) revealed several significantly upregulated malignancy-associated events (IL-6, JAK, STAT3, MTORC1, and KRAS signaling pathways) in the Sema4D high expression group as well as downregulation of p53 signaling (Fig. 5C). Sema4D was significantly associated with oncogenes RHOA, RRAS and immunomodulatory-related genes PTPRC and PLXNC1 (Fig. 5D). Pathway enrichment analysis revealed that Sema4D is involved in multiple immune-related pathways as well as the PI3K/AKT pathway (Fig. 5E). Numerous studies have demonstrated that excessive activation of PI3K/AKT signaling pathway is associated with excessive proliferation of tumor cells, inhibition of apoptosis and attenuation of the efficacy of immune checkpoint inhibitors (Zhang, Richmond & Yan, 2022). The expression of PI3K/AKT protein was explored by Western blot. p-PI3K/PI3K, p-AKT/AKT of Sema4D-shRNA group were decreased than those of Sema4D-NC group, which suggesting aberrant activation of the PI3K/AKT signaling pathway in the Sema4D-NC group (Fig. 5F).

DISCUSSION
Immunotherapy is one important method for melanoma treatment, when alone or in combination with anti-PD-1 and anti-PD-L1 may improve survival rates (Simeone, Grimaldi & Ascierto, 2015). Nivolumab is a PD-1 inhibitor with high affinity to PD-1 (Hirano et al., 2005). However, the ineffectiveness or resistance of anti-PD-1 to melanoma is a serious problem. In general, anti-PD-1 resistance are mainly derived into acquired resistance and innate resistance (Bridgeman et al., 2014;Marincola et al., 2000;Bronte et al., 2005;O'Donnell et al., 2017). The causes of anti-PD-1 or anti-PD-L1 resistance to melanoma are very complex and not fully understood. This study intends to elucidate that Sema4D may be involved in the sensitivity of melanoma to nivolumab therapy via PI3K/AKT signaling pathway. Nivolumab is a PD-1 inhibitor, PD-1 exists in T cells while PD-L1 exists in tumor cells, so the effect of nivolumab on tumor cells requires the presence of T cells. B16-F10 is a melanoma cell line derived from C57BL/6J mice, but tumor formation can also be inoculated subcutaneously in Balb/c mice (Colombo et al., 2019). In this experiment, Tumor Infiltrating Lymphocyte (TIL) was obtained from Balb/c mice and added to B16-F10 or B16-F10R cell to simulate tumor microenvironment. B16-F10 resistance cells were implanted subcutaneously in Balb/c mice by subcutaneous injection to observe tumor volume and weight.
Sema4D expressed on many cancer cells, Plexin-B1 also expression in cancer such as ovarian, melanoma, when Sema4D combined with Plexin-B1, it correlates with tumor immune infiltration, angiogenesis, and tumor progression in melanoma (Ch'ng & Kumanogoh, 2010;Lu et al., 2021). To understand the expression of Sema4D in melanoma resistance to anti-PD-1, B16-F10 resistant cell lines was established, after administered 50 ng/ml nivolumab, the killing ability of nivolumab was significantly weaker than that of B16-F10 cell lines, this means that resistant cell lines are established successfully. Then we explored mRNA and protein expression of Sema4D and Plexin-B1 in B16-F10 cell lines and B16-F10R cell lines, our results revealed that the mRNA and protein expression of Sema4D and Plexin-B1 in drug-resistant cell lines were overexpressed. Therefore, we provide the evidence for the first time that Sema4D and Plexin-B1 is related to anti-PD-1 resistance of melanoma.
To further analyzed the relationship between Sema4D and nivolumab, Sema4D were overexpression in B16-F10 cells and Sema4D expression was silencing by shRNA in B16-F10R cells. It was observed that overexpression of Sema4D can attenuates the therapeutic effect of nivolumab in B16-F10 cells and downregulation of Sema4D will increase the  (E) Pathway enrichment analysis showed that Sema4D is involved in the PI3K-AKT signaling pathway. (F) PI3K-AKT signaling pathway proteins expression after Sema4D knockdown. B16-F10R cells were derived into Sema4D-shRNA group and Sema4D-NC group, co-cultured with TIL at a 1:5 ratio and nivolumab 50 ng/ml, PI3K, AKT protein expression were detected. The experiment was repeated three times, n = 3. Western blotting showed that Sema4D was significantly associated with inhibition of PI3K-AKT signaling pathway . Compared with Sema4D-NC group, * * is p < 0.01. sensitivity of B16-F10R cell lines to nivolumab. In addition, we also investigated the effects of Sema4D knockdown on B16-F10R cells in response to nivolumab, downregulation of Sema4D also increased the inhibiting the growth of tumor cells and reducing cell invasion and migration. As reported by Rezaeepoor that silencing of Sema4D serve as a therapeutics target for the suppression of invasion, migration (Rezaeepoor et al., 2021). A study conducted by Rashidi illustrated silencing of Sema4D can elevated apoptosis rate of SW48 cells in response to 5-FU treatment (Rashidi et al., 2020). Moreover, we verified the enhancement of anti-PD-1 sensitivity by Sema4D knockdown in vivo xenograft models, mice treated with nivolumab after Sema4D knockdown showed significantly slower tumor growth. Taken together, these finding demonstrated that Sema4D deficiency can potentiates nivolumab treatment efficacy. We further analyzed the correlation between Sema4D and PD-L1 expression in anti-PD-1 resistance. It has been demonstrated that the presence of PD-L1 determines the response to anti-PD-1/PD-L1 therapy (Sunshine et al., 2017), regorafenib can promotes antitumor immunity via inhibiting PD-L1 expression in melanoma (Wu et al., 2019). JAK1/2 mutation leads to the lack of reactive PD-L1 expression, which is involved in the resistance of PD-1/PD-L1 inhibitors (Shin et al., 2017). In this study, we evaluated PD-L1 expression in B16-F10, B16-F10R cell and B16-F10R cell knockdown with Sema4D-shRNA, the results showed PD-L1 expression in B16-F10R was increased and Sema4D knockdown group is decrease, these suggest that the effects of Sema4D knockdown on Nivolumab in B16-F10R cell lines may be related to the decrease of PD-L1 expression. However, which way contributes to the PD-L1 downregulation after knockdown Sema4D remains unclear and needs further investigation.
PI3K is phosphorylated by binding to GRB2, and AKT phosphorylates target genes to regulate cell function (Pu et al., 2021). Sema4D and plexin-B1 is associated with osteosarcoma, in which PYK2-PI3K-Akt pathway is activated to promote tumor progression (Li et al., 2021). To explore the relationship between Sema4D and PI3K/AKT signaling pathways in anti-PD-1 resistance, we detected the expression of PI3K/AKT protein, in B16-F10R cell, p-PI3K/PI3K, p-AKT/AKT expression were significantly increased, when inhibited the Sema4D, p-PI3K/PI3K, p-AKT/AKT expression of Sema4D-shRNA group were decreased, these shown that Sema4D can regulate PI3K/AKT expression and activity. Bioinformatics analysis revealed that Sema4D is involved in the PI3K/AKT signaling pathway which is activated and plays important roles such as being involved in the development and occurrence in melanoma and it also activate receptor tyrosine kinases (Davies, 2012). However, whether the relationship between Sema4D and nivolumab